Researchers from the Kleckner Lab, in collaboration with researchers at CNRS in France and the Chinese Academy of Sciences, have found evidence that microscopic “inter-axis bridges,” which form between sister chromatids during mitosis, play several crucial roles throughout anaphase, the stage of cell division when the sister chromatids separate. Led by first author and MCB Research Associate Lingluo Chu, the team conducted a series of experiments to better understand how these bridges are removed when sister chromatids move to opposite poles and to determine whether bridges might play any unsuspected active role in that process. Their results appear in the journal PNAS (Proceedings of the National Academy of Science) PDF.
Most research on how chromosomes separate has focused on centromeres, where spindle fibers attach to specific protein complexes called kinetochores. Inter-axis bridges, by contrast, form all along the length of chromosome arms during late prophase and help keep sister chromatids lined up in parallel. Each chromatid comprises a linear array of chromatin loops emanating from a complex structural axis meshwork. Bridges link these axes and are made up of chromatin plus diverse structural components, including a protein called cohesin.
In all likelihood, bridges are built upon topological catenations between loops of sister chromatids. Previous work, motivated by the discovery of bridges, revealed that (contrary to 150 years of dogma), mitotic chromosomes are not helically coiled, but instead become shorter and fatter by modulation of their underlying loop/axis organization. Research further suggested that emergence of bridges and ensuing events are promoted by mechanical stress along chromosome axes.
The new paper by Chu et al describes anaphase sister separation as a three-step process. In the first step, termed “global separation,” the sister chromatids move apart along their lengths with concomitant elongation of bridges. In the second step, called “peeling apart,” the chromatids move further away from each other, starting from the centromere/kinetochore regions towards distal regions. At each peeling-apart “fork,” bridges are transiently stretched into extremely long and thin shapes, which are then removed through a process involving a DNA-detangling enzyme called Topoisomerase IIα. During the third step, residual catenations between the very ends of sister chromatids (called telomeres) are removed.
Each of the three steps appears to be driven by a different type of molecular separation force. During “global separation” release of cohesin allows the chromatin of sister chromatids to push one another apart. During the “peeling apart” step, the chromatids are tugged on by “poleward spindle forces”. And the final step occurs as the opposite poles of the dividing cells move farther apart, with resulting tension on residual connections proposed to trigger Topoisomerase IIα-mediated resolution of remaining inter-sister catenations.
Interestingly, adding a Topoisomerase IIα blocker to cells in the middle of “peeling apart” stopped the process “in its tracks”, raising the possibility that decatenation and/or removal of Topoisomerase IIα is/are the rate-limiting step in bridge disassembly. Kleckner and her colleagues argue that the spindle forces, which are moving sister chromatids apart, are working against resistance from bridges awaiting removal. This effect could concomitantly ensure not only that sister chromatids move synchronously towards their respective poles but, in addition, would keep them moving in a directed fashion without risk of discontinuous back-and-forth motion.
Future studies, being carried out in collaboration with SEAS postdoctoral fellow Hai-Yin Wu and MCB faculty Dan Needleman, will use laser ablation to further document chromatin pushing effects during global separation and to test the hypothesis that bridges are important for ensuring smooth, synchronous separation at anaphase.